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Background Human life is based on oxygen respiration and an enzymatic, free radical-dependent water chemistry, whose billions of parallel reactions take place at pH ∼7.4 and a temperature of 37°C, in accordance with the laws of chemistry. The cellular metabolic processes occur over time periods covered by the half-lives of ROS (reactive oxygen species) for °OH to over 10 s for LOS (lipid oxygen species), indicating that mixtures of free radicals form the basic components for these processes. Summary and Key Messages The main source of radicals is the mitochondrial conversion of 1-5 % oxygen into "primary" ROS and "secondary" LOS. Every endogenous and exogenous radical generation, triggered by "natural background radiation", "natural environment" or "solar radiation" leads to qualitatively similar mixtures of "primary" ROS and "secondary" LOS or RNS (reactive nitrogen species). A Multilevel Antioxidant Regulation, Repair and Protection System (MARRPS) keeps these radical mixtures in a steady state. Depending on the total number of free radicals, different areas of radical action are defined. The Free Radical Ground State (FRGS) with "homeostasis" and "adaptive homeostasis", the Free Radical Threshold Value (FRTV) and Free Radical Pathological Conditions (FRPC). The quantitative ratio ROS > LOS comprehensively characterizes the "homeostasis" and "adaptive homeostasis" area of the FRGS. The total number of free radicals cannot be measured directly in the "homeostasis" area. "Adaptive homeostasis" is achieved when excess radicals are stable produced beyond "homeostasis" of the FRGS. The quantity that remains controllable in this range is a maximum of ∼3.58 10¹² radicals/mg, the value of the body constant FRTV. The sensitized MARRPS provides "semi-stable homeostatic" states characterized by dual stability with ROS > LOS and a stable total ROS/LOS and RNS count beyond the basal FRGS "homeostasis". If the total number of all radicals exceeds the FRTV, where LOS > ROS, this initiates uncontrolled radical chain reactions. The partial failure of the MARRPS in the FRPC area leads to pathological processes which are the starting point for a hundred different diseases. The design principle described by this simple model applies universally to all aerobic life., (S. Karger AG, Basel.)

Cold atmospheric plasma (CAP) enables painless tissue treatment by producing reactive species including excited molecules and charged particles and is of great interest for medical applications. Medical CAP sources work in contact with air at ambient pressure, resulting in the generation of substantial amounts of reactive oxygen and nitrogen radicals. These radicals have a significant influence on cellular biochemistry, are crucial components of the immune system, and play a central role in wound therapy. CAP has a variety of applications, with a particular emphasis on tissue treatment in dermatology. It eradicates microorganisms by preventing biofilm formation so that wounds can be effectively disinfected and treated antiseptically. Using both in vitro and ex vivo methods, a comprehensive preclinical assessment of a novel battery-operated cold plasma handheld device with a reusable, and autoclavable glass cylinder was performed. The objectives were to evaluate the potential impact of single CAP application on radical formation with and without wound dressing, by directly measuring radicals in skin, to investigate the influence of CAP application on antimicrobial activity and cytotoxicity in vitro, and to assess skin tolerance ex vivo. The direct effect of CAP on the formation of radicals in the skin after plasma application at different levels with and without wound dressing was demonstrated quantitatively for the first time using electron paramagnetic resonance spectroscopy. Free radicals were measured in the skin as a function of the duration of CAP treatment. Furthermore, it was found that an alginate or wound plaster dressing does not significantly inhibit radical formation in skin compared to application without a dressing. In vitro and ex vivo data showed no cytotoxic potential with simultaneous efficacy against bacteria strains and no risk of temperature rise, pH change, skin barrier or DNA damage. These results show a high potential for wound healing applications in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The concentration of air pollution is gradually increasing every year so that daily skin exposure is unavoidable. Dietary supplements and topical formulations currently represent the protective strategies to guard against the effects of air pollution on the body and the skin. Unfortunately, there are not yet enough methods available to measure the effectiveness of anti-pollution products on skin. Here, we present two ex vivo methods for measuring the protective effect against air pollution of different cream formulations on the skin: Electron paramagnetic resonance (EPR) spectroscopy and autofluorescence excited by 785 nm using a confocal Raman microspectrometer (CRM). Smoke from one cigarette was used as a model pollutant. EPR spectroscopy enables the direct measurement of free radicals in excised porcine skin after smoke exposure. The autofluorescence in the skin was measured ex vivo, which is an indicator of oxidative stress. Two antioxidants and a chelating agent in a base formulation and a commercial product containing an antioxidant mixture were investigated. The ex vivo studies show that the antioxidant epigallocatechin-3-gallate (EGCG) in the base cream formulation provided the best protection against oxidative stress from smoke exposure for both methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin's response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm 2 and a UV lamp at 3 mJ/cm 2 . Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana-Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes., (© 2024. The Author(s).)

Excessive exposure to ultraviolet (UV) light leads to acute and chronic UV damage and is the main risk factor for the development of skin cancer. In most countries with western lifestyle, the topical application of sunscreens on UV-exposed skin areas is by far the most frequently used preventive measure against sunburn. Further than preventing sunburns, increasing numbers of consumers are appreciating sunscreens with a medium- to high-level sun protective factor (SPF) as basis for sustainable-skin ageing or skin cancer prevention programs. However, recent investigations indicate that clinically significant DNA damages as well as a lasting impairment of cutaneous immunosurveillance already occur far below the standard of one minimal erythema dose (MED) sunburn level, which contributes to the current discussion of the clinical value of high-protective SPF values. Ex vivo investigations on human skin showed that the application of SPF30 reduces DNA damage for a day long sun exposure (24 MED) drastically by about 53% but is significantly surpassed by SPF100 reducing DNA damage by approx. 73%. Further analysis on different SPF protection levels in UV-exposed cell culture assays focusing on IL-18, cell vitality and cis/trans-urocanic acid support these findings. Whereas SPF30 and SPF50 + sunscreens already offer a solid UVB cover for most indications, our results indicate that SPF100 provides significant additional protection against mutagenic (non-apoptotic-) DNA damage and functional impairment of the cutaneous immunosurveillance and therefore qualifies as an optimized sunscreen for specifically vulnerable patient groups such as immunosuppressed patients, or skin cancer patients., (© 2024 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

The inactivation of multi resistant pathogens is an important clinical need. One approach is UV-C irradiation, which was previously not possible in vivo due to cytotoxicity. Recently, far UV-C irradiation at λ < 240 nm was successfully used on skin with negligible damage. A potential application site is the nasal vestibule, where MRSA accumulates and cannot be treated using antiseptics. We irradiated 3D mucosa models and excised human mucosa with 222 and 233 nm far UV-C in comparison to 254 nm and broadband UV-B. Eradication efficiency was evaluated by counting colony forming units; irritation potential was evaluated by hen's egg-chorioallantoic membrane assay and trans epithelial electrical resistance; cell viability was assessed by MTT. DNA damage and cell protective mechanisms were evaluated immunohistopathologically. On mucosa models, MRSA reduced by ≈ 5 log 10 for 60 mJ/cm 2 irradiation at 233 nm. A slightly increased cell viability was observed after 24 h. Lower doses showed lower irritation potential than the positive controls or commercial mouthwash, while 80 mJ/cm 2 had strong irritation potential. DNA damage occurred only superficially and decreased after 24 h. On excised human mucosa, < 10% of keratinocytes were affected after 150 mJ/cm 2 222 nm or 60 mJ/cm 2 233 nm., (© 2023. The Author(s).)

Far-UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far-UVC-induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far-UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer-positive (CPD + ) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type-dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm 2 . As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two-photon excited fluorescence lifetime imaging (TPE-FLIM) finding a higher ratio up to skin type IV-V. As far-UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far-UVC compared to UVB., (© 2023 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm -2 as reported recently. In addition, only minor epidermal DNA lesions were observed in ex vivo human skin and in vitro epidermal models <10% of the minimal erythema dose of UVB radiation. To broaden the potential range of applications of such systems, e.g. to include postoperative application on wounds for the purpose of decontamination, we assessed how a disruption of normal anatomic skin structure and function influences the skin damage induced by light from 233 nm far-UVC LEDs. Thus, we induced superficial skin wounds by mechanical detachment of the stratum corneum in ex vivo human skin. Barrier-disruption of the skin could be successfully determined by measuring an increase in the transepidermal water loss (TEWL) and the stratum corneum loss could be determined morphologically by 2-photon microscopy (2-PM). After far-UVC irradiation of the skin, we screened the tissue for the development of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). The abundance of DNA lesions was elevated in wound skin in comparison to intact skin after irradiation with far-UVC. However, no increase in DNA lesions was detected when artificial wound exudate consisting of cell culture medium and serum was applied to the disrupted skin surface prior to irradiation. This effect agrees with the results of ray tracing simulations of the absorption of far-UVC light incident on a superficial skin wound. Interestingly, no significant deviations in radical formation between intact skin and superficially wounded skin were detected after far-UVC irradiation as analyzed by electron paramagnetic resonance (EPR) spectroscopy. In conclusion, 233 nm LED light at a dose of 60 mJ/cm 2 could be applied safely on superficial wounds for the purpose of skin antisepsis as long as the wounds are covered with wound fluid., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized.

Fungal infections have increased considerably over the last decades, becoming progressively resistant to common drugs. UVC light has shown microbiological eradication effects, whereby the wavelength of 254 nm is strongly carcino- and mutagenic. Therefore, 222 and 233 nm, which do not significantly harm skin cells, were tested for their antifungal effects. Microbicidal doses were reached at 40 mJ/cm 2 for both wavelengths, resulting in only minor superficial skin damage (<20 μm). UVC irradiation with far-UVC <240 nm represents a new opportunity to effectively eradicate even larger pathogens on tissue causing no or strongly reduced DNA and tissue damage., (© 2022 The Authors. Mycoses published by Wiley-VCH GmbH.)

Oxidative stress as a driver of disease is reinforcing the trend towards supplementation with antioxidants. While antioxidants positively influence the redox status when applied at physiological doses, higher concentrations may have pro-oxidative effects. Precise assessment methods for testing the supply of antioxidants are lacking. Using in-situ-irradiation as stressor and electron paramagnetic resonance (EPR) spectroscopy as readout system for formed radicals, a stress response assessment method was developed, using protein solutions and plasma samples from transfusion medicine. The method was validated in a double-blind placebo-controlled in vivo cross-over pilot study in blood plasma samples of individuals before and after vitamin C supplementation. Reference measurements were performed for the exogenous antioxidants β-carotene and vitamin C, and glutathione as an endogenous representative. Malondialdehyde was studied for oxidative stress indication. Protein solutions without antioxidants showed a linear increase in radical concentration during irradiation. The in-vitro-addition of vitamin C or plasma samples from subjects displayed two slopes (m 1 , m 2 ) for radical production, whereby m 1 represented the amount of antioxidants and proteins, m 2 only the protein content. These two slopes in combination with the intervening transition area (T) were used to calculate the oxidative stress coping capacity (OSC), which correlated positively with vitamin C concentration in blood plasma, while oxidative stress biomarkers showed only fluctuations within their reference ranges. Furthermore, a selective radical quenching mechanism for vitamin C was observed: the proportion of reactive oxygen species (ROS) in the plasma samples was degraded in dependence to the vitamin C concentration ingested. The proportion of lipid oxygen species (LOS) remained stable while the ascorbyl radical increased with higher vitamin C intake. OSC may represent a sensitive method to detect treatment effects on the redox status in vivo in future validation and treatment studies, and potentially in clinical routine., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)-the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed., Competing Interests: The authors declare no conflict of interest.

A newly developed UVC LED source with an emission wavelength of 233 nm was proved on bactericidal efficacy and skin tolerability. The bactericidal efficacy was qualitatively analysed using blood agar test. Subsequently, quantitative analyses were performed on germ carrier tests using the MRSA strain DSM11822, the MSSA strain DSM799, S. epidermidis DSM1798 with various soil loads. Additionally, the compatibility of the germicidal radiation doses on excised human skin and reconstructed human epidermis was proved. Cell viability, DNA damage and production of radicals were assessed in comparison to typical UVC radiation from discharge lamps (222 nm, 254 nm) and UVB (280-380 nm) radiation for clinical assessment. At a dose of 40 mJ/cm 2 , the 233 nm light source reduced the viable microorganisms by a log 10 reduction (LR) of 5 log 10 levels if no soil load was present. Mucin and protein containing soil loads diminished the effect to an LR of 1.5-3.3. A salt solution representing artificial sweat (pH 8.4) had only minor effects on the reduction. The viability of the skin models was not reduced and the DNA damage was far below the damage evoked by 0.1 UVB minimal erythema dose, which can be regarded as safe. Furthermore, the induced damage vanished after 24 h. Irradiation on four consecutive days also did not evoke DNA damage. The radical formation was far lower than 20 min outdoor visible light would cause, which is classified as low radical load and can be compensated by the antioxidant defence system., (© 2022. The Author(s).)

The exposure to sun radiation is indispensable to our health; however, a long-term and high exposure could lead to cell damage, erythema, premature skin aging, and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals which may induce oxidative stress at elevated concentrations. Different skin models, such as porcine-, murine-, human- ex vivo skin, reconstructed human skin (RHS) and human skin in vivo, were investigated during and after irradiation using X- and L-band EPR spectroscopy within different spectral regions (UVC to NIR). The amount of radical formation was quantified with the spin probe PCA and the radical types were measured ex vivo with the spin trap DMPO. The radiation dose influences the types of radicals formed in the skin. While reactive oxygen species (ROS) are always pronounced at low doses, there is an increase in lipid oxygen species (LOS) at high doses. Furthermore, the radical types arise independent from the irradiation wavelength, whereas the general amount of radical formation differs with the irradiation wavelength. Heat pre-stressed porcine skin already starts with higher LOS values. Thus, the radical type ratio might be an indicator of stress and the reversal of ROS/LOS constitutes the point where positive stress turns into negative stress.Compared to light skin types, darker types produce less radicals in the ultraviolet, similar amounts in the visible and higher ones in the infrared spectral region, rendering skin type-specific sun protection a necessity., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2021.)

Most modern sunscreens contain physical filters, which scatter the sunlight, increasing the photons' pathway in the upper stratum corneum. This effect can lead to a better efficacy of the UV filters and improve the diffuse reflection. However, the addition of nanosized inorganic UV filters reduces the antioxidant capacity of sunscreen formulations. Two cream formulations (F1, F2) which differ in the ingredient PEG75 Lanolin (F2), have been characterized for their radical protection factor (RPF) and their optical properties in vitro using electron paramagnetic resonance (EPR) spectroscopy and UV/VIS spectrometry. The RPF for PEG-75 Lanolin was also determined. Furthermore, their radical protection properties were analyzed on porcine skin ex vivo after visible light irradiation by EPR. The structure of each formulation in the skin surface was determined by reflectance confocal microscopy in vivo. The addition of lanolin increased the reflectance and reduced the transmittance for visible light, improving the scattering drastically. Besides, the antioxidant capacity was also increased for F2, something unpublished until now. F1 presented a lower scattering provided by starches. The sunscreens showed high scattering properties and antioxidant capacity, especially for F2, which presented the lowest radical formation in the skin model. These results are consistent with the RPF measurements where F2 has a higher RPF value (193 ± 3 × 10 14 radicals/mg) than F1 (155 ± 4 × 10 14 radicals/mg) and for PEG-75 Lanolin (37 ± 1 × 10 14 radicals/mg). The combination of starches and PEG-75 Lanolin is the first solution to provide both, light scattering and antioxidant capacity, in sunscreens., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Multiresistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause serious postoperative infections. A skin tolerant far-UVC (< 240 nm) irradiation system for their inactivation is presented here. It uses UVC LEDs in combination with a spectral filter and provides a peak wavelength of 233 nm, with a full width at half maximum of 12 nm, and an irradiance of 44 µW/cm 2 . MRSA bacteria in different concentrations on blood agar plates were inactivated with irradiation doses in the range of 15-40 mJ/cm 2 . Porcine skin irradiated with a dose of 40 mJ/cm 2 at 233 nm showed only 3.7% CPD and 2.3% 6-4PP DNA damage. Corresponding irradiation at 254 nm caused 15-30 times higher damage. Thus, the skin damage caused by the disinfectant doses is so small that it can be expected to be compensated by the skin's natural repair mechanisms. LED-based far-UVC lamps could therefore soon be used in everyday clinical practice to eradicate multiresistant pathogens directly on humans., (© 2021. The Author(s).)

Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label-mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof-of-principle penetration experiments in human skin ex vivo., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The topical photodynamic therapy (PDT) is mainly used in the treatment of dermato-oncological diseases. The distribution and functionality of the photosensitizer Tetrahydroporphyrin-Tetratosylat (THPTS) was investigated using microscopic and spectroscopic methods after topical application to excised porcine skin followed by irradiation. The distribution of THPTS was determined by two-photon tomography combined with fluorescence lifetime imaging (TPT/FLIM) and confocal Raman microspectroscopy (CRM). The radicals were quantified and characterized by electron paramagnetic resonance (EPR) spectroscopy. Results show a penetration depth of THPTS into the skin down to around 12 ± 5 µm. A penetration of THPTS through the stratum corneum was not clearly observable after 1 h penetration time, but cannot be excluded. The irradiation within the phototherapeutic window (spectral range of visible and near infrared light in the range ≈ 650-850 nm) is needed to activate THPTS. An incubation time of 10 min showed the highest radical production. A longer incubation time affected the functionality of THPTS, whereby significant less radicals were detectable. During PDT mainly reactive oxygen species (ROS) and lipid oxygen species (LOS) are produced. Overall, the irradiation dose per se influences the radical types formed in skin. While ROS are always prominent at low doses, LOS increase at high doses, independent of previous skin treatment and the irradiation wavelength used., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The sun protection factor (SPF) is related to the selected UV filters. The objective of this study was to evaluate and compare the rheological behavior and texture profile of two sunscreen formulations and to correlate these data with the obtained SPF values. Two formulations (F1 and F2) were developed with the same type and amount of UV filters - whereby one of them also contained ethoxylated lanolin as additional film former (F2). Their rheological behavior, texture profile and in vitro and in vivo SPF were analyzed. The film-forming properties were evaluated by skin profilometry and diffuse reflectance spectroscopy. The structures of the formulations were examined by two-photon tomography combined with fluorescence lifetime imaging, and the penetration profile into the stratum corneum was investigated by tape stripping. The formulation with lanolin presented lower and constant values for physical-mechanical parameters, with a higher and better reproducible SPF. Both formulations did not penetrate the viable epidermis. In conclusion, formulations with better surface deposition on the skin surface can influence the film formation and, consequently, improve the SPF. These findings are important to improve the efficacy of sunscreen formulations and reduce the addition of UV filters., (Copyright © 2021. Published by Elsevier B.V.)

Ultraviolet (UV) radiation leads to the formation of free radicals, which may cause immunological modulations, skin aging or skin cancer. Sunlight exposure in the UVA region according to CIE 85 promotes almost 46% of radical formation in skin. A critical radical concentration characterized by the inversion of the domination of primary ROS (reactive oxygen species) to an excess of secondary LOS (lipid oxygen species) is proven for the spectral regions UV and or VIS light and is intended to be a marker for an imbalance in the redox system, which can no longer compensate harmful effects. To investigate whether this transition point is also universally valid for one spectral region, the radical formation during and after targeted UVA in situ-irradiation at 365 ± 5 nm and three different irradiances (31, 94 and 244 mW/cm 2 ) was investigated in ex vivo porcine skin using x-band electron paramagnetic resonance (EPR) spectroscopy. The quantification was performed with the spin probe 3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl (PCA), the spin trap 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO) was used to characterize the radical species. Furthermore, the viability of the skin cells after irradiation was controlled by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, skin integrity was examined by histological analysis. A significant dose dependence in the radical formation is given at higher irradiance. The transition point was detected in the range of 0.5 MED after irradiation with the highest irradiance. From this point on the proportion of LOS increases with increasing dose and the proportion of ROS decreases. After switching off the UVA irradiation no further quantitative changes were detected, but rapid changes in the radical pattern were observed demonstrating the importance of in situ irradiation during the use of spin traps. Heat-pre-stressed skin showed more LOS than ROS already at the beginning of the irradiation, leading to the assumption that the transition point to the distress-level has already been reached. In summary, a postulated transition point could be verified for the UVA spectral region using only one spin trap combined with in-situ irradiation. A certain degree of stress is necessary to detect an inversion of the ratio of ROS to LOS. This reversal indicates an imbalance in the redox status. However, at low intensities no changes at all in radical pattern appeared over time (dose), probably it can be compensated by adaptation processes of the skin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Electron paramagnetic resonance (EPR) spectroscopy is an established method for the measurement of free radicals. Solar radiation is essential for human life as it stimulates vitamin D synthesis and well-being. However, an excessive dose of solar radiation leads to the formation of free radicals. Here, we describe an EPR method for measuring the amount of radicals induced by UVA irradiation in excised skin. For the first time, a wavelength stable UVA LED (365 nm) was used. The method allows the quantitative determination of radicals in skin before, during, and after UVA irradiation. A dose-dependent radical production could be demonstrated, independent of the yielded power.

The daily consumption of tobacco products leads to a boost in free radical production in tissues, promoting the risk for malignancies, metabolic alterations and chronic-inflammatory diseases. This study aimed to broaden the knowledge of the status of the antioxidative (AO) system in the skin, compared to the blood, of healthy appearing smokers. Both, the basic status compared to non-smokers and the short-term impact of controlled cigarette consumption in smokers were analyzed. Our study showed that the basic level of the AO system of smokers significantly differed from that of non-smokers. As determined by resonant Raman spectroscopy (RRS), the levels of exogenous AOs were decreased in both, the skin, in vivo (β-carotene and lycopene), and blood plasma (β-carotene only). In contrast, the levels of glutathione (GSH), the prototypical endogenous AO, which were analyzed by fluorimetric assays in cutaneous tape strips and blood plasma, were increased in the skin, although unchanged in the blood of smokers. Elevated cutaneous GSH levels were reflected by an elevated overall radical scavenging activity in the skin, as quantified by non-invasive electron paramagnetic resonance (EPR) spectroscopy. Analysis of the expression of selected stress-associated genes in blood immune cells by quantitative RT-PCR in subgroups of non-smokers and smokers additionally demonstrated the downregulation of AKR1C2 in smokers, and its negative correlation with blood plasma levels of the protective immune mediator interleukin-22, assessed by the ELISA technique. Controlled cigarette consumption did not alter exogenous or endogenous AOs in the skin of smokers, but decreased lycopene levels in blood plasma. Moreover, there was a decline in blood IL-22 levels, while no relevant response of blood cell gene expressions was found after the considered short time. Our data therefore demonstrate a strengthened endogenous AO status in the skin of smokers, which may indicate a long-term adaptation to chronic oxidative stress in this specific organ. While this effect was not clearly visible in the blood, this compartment seems to be useful as an immediate indicator of the body's AO consumption. Moreover, decreased levels of AKR1C2, which we show for the first time to be expressed in immune cells, may be a candidate marker for long-term smoking. In addition, this study demonstrates that the rate constant of a spin probe decline determined by EPR spectroscopy mainly represents the endogenous AO status of a tissue., Competing Interests: The authors declare no conflict of interest.

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications.

Electron paramagnetic resonance (EPR) spectroscopy represents an established tool to study properties of microenvironments, e.g. to investigate the structure and dynamics of biological and artificial membranes. In this study, the partitioning of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in ex vivo human abdominal and breast skin, ex vivo porcine abdominal and ear skin as well as normal and inflammatory in vitro skin equivalents was investigated by EPR spectroscopy. Furthermore, the stratum corneum (SC) lipid composition (as determined by high-performance thin-layer chromatography), SC lipid chain order (probed by infrared spectroscopy) and the SC thickness (investigated by histology) were determined in the skin models. X-band EPR measurements have shown that TEMPO partitions in the lipophilic and hydrophilic microenvironment in varying ratios in different ex vivo and in vitro skin models. Ex vivo human abdominal skin exhibited the highest amount of TEMPO in the lipophilic microenvironment. In contrast, the lowest amount of TEMPO in the lipophilic microenvironment was determined in ex vivo human breast skin and the inflammatory in vitro skin equivalents. Individual EPR spectra of epidermis including SC and dermis indicated that the lipophilic microenvironment of TEMPO mainly corresponds to the most lipophilic part of the epidermis, the SC. The amount of TEMPO in the lipophilic microenvironment was independent of the SC lipid composition and the SC lipid chain order but correlated with the SC thickness. In conclusion, EPR spectroscopy could be a novel technique to determine differences in the SC thickness, thus suitably complementing existing methods., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis is still not fully understood. Since inflammatory processes correlate with oxidative stress, the redox status may play a key role in AD. In this study, electron paramagnetic resonance (EPR) spectroscopy was mainly used to investigate the redox status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro using EPR spin probes (TEMPO, PCA) and a spin trap (DMPO). The total antioxidant status in the hydrophilic and lipophilic compartments of skin (microenvironment) showed no differences between the skin equivalents. In the inflammatory skin equivalents, a decreased glutathione concentration in the epidermis and an increased metabolic radical production could be observed compared to normal skin equivalents. The induction of external stress by simulated solar irradiation (UVB-NIR) resulted in the same amount and type of radicals in normal and inflammatory skin equivalents. For the first time, the antioxidant and oxidant status of inflammatory in vitro skin equivalents was analyzed by EPR to elucidate their redox status using different methods which focus on various microenvironments. Our investigations suggested that the redox status in atopic skin could be different, but this should be investigated more comprehensively, because the results can vary depending on the used methods and where the investigations take place., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nanoparticles (NPs) are promising carriers for dermal and transdermal drug delivery. However, the underlying dynamics of drug release from the NPs, especially, how the physiological changes of diseased skin influence the drug release, remain poorly understood. We utilized electron paramagnetic resonance (EPR) and confocal laser scanning microscopy (CLSM) to comprehensively investigate the penetration behavior of a spin-labeled dexamethasone (DxPCA)-loaded pH-sensitive Eudragit® L 100 NP on intact and barrier-disrupted skins. The EPR investigation showed that a rapid in vitro DxPCA release from the NPs was triggered above pH 5.9. It also demonstrated that the NPs considerably improved the cutaneous penetration of the model drug in comparison to a commercial cream. Besides, as compared to the intact skin, a faster drug release and a higher drug penetration into the viable skin layers were obtained with barrier-disrupted skin. In accordance, CLSM studies confirmed that the NPs enhanced the penetration of the lipophilic model drug Nile red (NR) across the skin, whose penetration depth into glabrous skin was 160 μm. Moreover, a significant transfollicular penetration of NR from the NPs was observed. In conclusion, the pH-sensitive Eudragit® L 100 NPs improved the cutaneous penetration and controlled the release of a lipophilic drug, especially on barrier-disrupted skin. This may allow targeted drug delivery to lesional skin, avoiding side effects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Objectives: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long., Material and Methods: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively., Results: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro., Conclusion: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The skin is exposed to many stress factors which, in turn, can promote a shift of the antioxidant (AO) network towards the prooxidative side, supporting the development of various skin disorders. A balanced diet, in combination with a healthy lifestyle could reduce oxidative stress. Carotenoids are essential nonenzymatic AOs and main components of the exogenous AO system. To examine the interdependence between endogenous and exogenous AOs, secondary keratinocytes (HaCaT) were treated with various Beta (β-)-carotene concentrations with subsequent stress treatment by moderate irradiation (700-2000 nm). To facilitate the uptake of β-carotene, an innovative nanocrystal solution was used. Cell viability assay was applied to HaCaT cells to evaluate suitable concentration of β-carotene, whereby the uptake was measured by resonant Raman spectroscopy. The redox status was determined before and after supplementation with two selected β-carotene concentrations (0.02 and 0.1 μg/ml) and irradiation. Reactive oxygen species (ROS) were measured by electron paramagnetic resonance spectroscopy and the AO glutathione (GSH) by a fluorescent-based assay for evaluating the endogenous redox status. An increase of ROS and a reduction of GSH after irradiation was observed. Interestingly, the applied β-carotene, already induce oxidative stress. Nevertheless, an effective protection against irradiation could be observed for the lower dose. The high dose turned pro-oxidative., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, β-cyclodextrin (βCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of βCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of βCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in βCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.

Exogenous factors can cause an imbalance in the redox state of biological systems, promoting the development of oxidative stress, especially reactive oxygen species (ROS). To monitor the intensity of ROS production in secondary keratinocytes (HaCaT) by diesel exhaust particles and thermoresponsive nanogels (tNG), electron paramagnetic resonance (EPR) spectroscopy after 1 and 24 h of incubation, respectively, was applied. Their cytotoxicity was analyzed by a cell viability assay (XTT). For tNG an increase in the cell viability and ROS production of 10% was visible after 24 h, whereas 1 h showed no effect. A ten times lower concentration of diesel exhaust particles exhibited no significant toxic effects on HaCaT cells for both incubation times, thus normal adult human keratinocytes (NHK) were additionally analyzed by XTT and EPR spectroscopy. Here, after 24 h a slight increase of 18% in metabolic activity was observed. However, this effect could not be explained by the ROS formation. A slight increase in the ROS production was only visible after 1 h of incubation time for HaCaT (9%) and NHK (14%).

Background: A nutrition rich in fruit and vegetables and a healthy lifestyle become more and more important in the industrial countries to counteract oxidative stress and promote health. For many years, it has been possible to control human cutaneous carotenoids noninvasively by resonance Raman spectroscopic systems and by spatially resolved reflectance spectroscopy., Methods: Ten volunteers took a commercially available fruit and vegetable extract daily for a time period of 5 weeks. A second group served as control group and did not take any supplements (10 volunteers). To monitor the status of the cutaneous carotenoids noninvasively, an optical sensor based on multiple spatially resolved reflectance spectroscopy was applied once a week., Results: The study could demonstrate that the intake of the supplement significantly increase the cutaneous carotenoid values of the young adults by 50%. The control group without any supplementation showed also significantly increased values, ie, by 10%, which might be due to the fact that their lifestyle was controlled., Conclusion: The results illustrate that a biofeedback by measuring the skin carotenoids could improve the lifestyle of young adults and that a regular consumption of fruit and vegetables directly or as a drink can increase the concentration of cutaneous carotenoids significantly., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer-based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core-multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual-frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) is associated with the CMS. The micro-environment of the drug loaded to CMS nanoparticles was investigated by pulsed high-field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g-, A-matrices, and spin-lattice relaxation time) represent specific probes for the micro-environment. Additionally, the rotational correlation time of spin-labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG&#95;dPG&#95;tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG&#95;dPG&#95;pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.

Background/aim: The main reason for extrinsic skin aging is the negative action of free radicals. The formation of free radicals in the skin has been associated with ultraviolet (UV) exposure and also to visible (VIS) and near-infrared (NIR) irradiations. The aim of the present study was to evaluate the efficacy of a sunscreen in the whole solar range., Methods: The radical-scavenging activity of a sunscreen in the UV, VIS, and NIR ranges was evaluated using electron paramagnetic resonance spectroscopy. Ex vivo penetration profiles were determined using confocal Raman microscopy on porcine ear skin at different time points after application., Results: Compared to the untreated skin, the sunscreen decreased the skin radical formation in the UV and VIS regions. Additional protection in the VIS and NIR ranges was observed for the sunscreen containing antioxidants (AO). The penetration depth of the cream was less than 11.2 ± 3.0 µm for all time points., Conclusion: A sunscreen containing AO improved the photoprotection in the VIS and NIR ranges. The sunscreen was retained in the stratum corneum. Therefore, these results show the possibility of the development of effective and safer sunscreen products., (© 2017 S. Karger AG, Basel.)

The targeted design of nanoparticles for efficient drug loading and defined release profiles is even after 25years of research on lipid-based nanoparticles still no routine procedure. It requires detailed knowledge about the interaction of the drug with the lipid compounds and about its localisation and distribution in the nanoparticle. We present here an investigation on nano-sized lipid particles (NLP) composed of Gelucire and Witepsol as solid lipids, and Capryol as liquid lipid, loaded with Dexamethasone, a glucocorticoid used in topical treatment of inflammatory dermal diseases. The interactions of Dexamethasone, which was spin-labelled by 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (DxPCA), with its microenvironment are monitored by EPR spectroscopy at 94GHz at low temperatures. The mobility of the spin-labelled drug was probed by X-band EPR at room temperature. In order to relate the magnetic and dynamic parameters deduced from EPR to the local environment of the spin probe in the NLP, investigations of DxPCA in the individual lipid compounds were carried out. The magnetic parameters reflecting the polarity of DxPCA's environment as well as the parameters describing the mobility of the drug reveal that in the case of colloidal dispersions of the lipids and also the NLP DxPCA is attached to the surface of the nanoparticles. Although the lipophilic drug is almost exclusively associated with the NLP in aqueous solution, dilution experiments show, that it can be easily released from the nanoparticle., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the last decade, cutaneous carotenoid measurements have become increasingly popular, as carotenoids were found to be a biomarker of nutrition rich in fruits and vegetables, permitting monitoring of the influence of various stress factors. For such measurements, in addition to the specific and selective resonance Raman spectroscopy (RRS), newly developed low expensive small and mobile sensors that are based on spatially resolved reflectance spectroscopy (SRRS) are used for cutaneous carotenoid measurements. Human volunteers of different age exhibiting skin types I to III were investigated using RRS and two SRRS-based sensors to determine the influence of these parameters on the measuring results. In two studies on volunteers of either the same age or skin type, however, the respective other parameter being varied and no significant influences of age or skin type could be detected. Furthermore, the kinetic changes resulting from the intake and discontinued intake of a vegetable extract rich in carotenoids showed a good correlation among the three sensors and with the detected blood carotenoids. This illustrates that the SRRS-based sensors and RRS device provide reliable cutaneous carotenoid values independent of age and skin types I to III of the volunteers.

Sunlight represents an exogenous factor stimulating formation of free radicals which can induce cell damage. To assess the effect of the different spectral solar regions on the development of free radicals in skin, in vivo electron paramagnetic resonance (EPR) investigations with human volunteers and ex vivo studies on excised human and porcine skin were carried out. For all skin probes, the ultraviolet (UV) spectral region stimulates the most intensive radical formation, followed by the visible (VIS) and the near infrared (NIR) regions. A comparison between the different skin models shows that for UV light, the fastest and highest production of free radicals could be detected in vivo, followed by excised porcine and human skin. The same distribution pattern was found for the VIS/NIR spectral regions, whereby the differences in radical formation between in vivo and ex vivo were less pronounced. An analysis of lipid composition in vivo before and after exposure to UV light clearly showed modifications in several skin lipid components; a decrease of ceramide subclass [AP2] and an increase of ceramide subclass [NP2], sodium cholesterol sulphate and squalene (SQ) were detectable. In contrast, VIS/NIR irradiation led to an increase of ceramides [AP2] and SCS, and a decrease of SQ. These results, which are largely comparable for the different skin models investigated in vivo and ex vivo, indicate that radiation exposure in different spectral regions strongly influences radical production in skin and also results in changes in skin lipid composition, which is essential for barrier function., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dendritic core-multi shell (CMS) particles are polymer based systems consisting of a dendritic polar polyglycerol polymer core surrounded by a two-layer shell of nonpolar C18 alkyl chains and hydrophilic polyethylene glycol. Belonging to nanotransport systems (NTS) they allow the transport and storage of molecules with different chemical characters. Their amphipihilic character CMS-NTS permits good solubility in aqueous and organic solutions. We showed by multifrequency electron paramagnetic resonance (EPR) spectroscopy that spin-labeled 5-doxyl stearic acid (5DSA) can be loaded into the CMS-NTS. Furthermore, the release of 5DSA from the carrier into the stratum corneum of porcine skin was monitored ex vivo by EPR spectroscopy. Additionally, the penetration of the CMS-NTS into the skin was analyzed by fluorescence microscopy using indocarbocyanine (ICC) covalently bound to the nanocarrier. Thereby, no transport into the viable skin was observed, whereas the CMS-NTS had penetrated into the hair follicles down to a depth of 340 μm ± 82 μm. Thus, it could be shown that the combined application of fluorescence microscopy and multi-frequency EPR spectroscopy can be an efficient tool for investigating the loading of spin labeled drugs to nanocarrier systems, drug release and penetration into the skin as well as the localization of the NTS in the skin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

UV irradiation is one of the most harmful exogenous factors for the human skin. In addition to the development of erythema, free radicals, that is reactive oxygen species (ROS), are induced under its influence and promote the development of oxidative stress in the skin. Several techniques are available for determining the effect of UV irradiation. Resonance Raman spectroscopy (RRS) measures the reduction of the carotenoid concentration, while electron paramagnetic resonance (EPR) spectroscopy enables the analysis of the production of free radicals. Depending on the method, the skin parameters are analysed in vivo or ex vivo. This study provides a critical comparison between in vivo and ex vivo investigations on the ROS formation and carotenoid depletion caused by UV irradiation in human skin. The oxygen content of tissue was also determined. It was shown that the antioxidant status measured in the skin samples in vivo and ex vivo was different. The depletion in the carotenoid concentration in vivo exceeded the value determined ex vivo by a factor of about 1.5, and the radical formation after UV irradiation was significantly greater in vivo by a factor of 3.5 than that measured in excised human skin, which can be explained by the lack of oxygen ex vivo., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

UV irradiation leads to the formation of reactive oxygen species (ROS). An imbalance between the antioxidant system and ROS can lead to cell damage, premature skin aging or skin cancer. To counteract these processes, antioxidants such as coenzyme Q10 (CoQ10) are contained in many cosmetics. To improve and optimize cell/tissue penetration properties of the lipophilic CoQ10, ultra-small lipid nanoparticles (usNLC) were developed. The antioxidant effectiveness of CoQ10-loaded usNLC compared to conventional nanocarriers was investigated in the human keratinocyte cell line HaCaT. Using confocal laser scanning microscopy investigations of the carriers additionally loaded with nile red showed a clear uptake into cells and their distribution within the cytoplasm. By use of the XTT cell viability test, CoQ10 concentrations of 10-50 μg/ml were shown to be non-toxic, and the antioxidant potential of 10 μg/ml CoQ10 loaded usNLC in the HaCaT cells was analyzed via electron paramagnetic resonance spectroscopy after cellular exposure to UVA (1J/cm(2)) and UVB (18 mJ/cm(2)) irradiation. In comparison with the CoQ10-loaded conventional carriers, usNLC-CoQ10 demonstrated the strongest reduction of the radical formation; reaching up to 23% compared to control cells without nanocarrier treatment. Therefore, usNLC-CoQ10 are very suitable to increase the antioxidant potential of skin., (Copyright © 2014 Elsevier B.V. All rights reserved.)